Host cellular genes regulations during H5N1 Avian Influenza virus infection and their protein-protein interactions

2017-02-23T04:03:05Z (GMT) by RMT Balasubramaniam, Vinod
Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and replicates in high levels in the respiratory tract especially in the lungs of poultry inducing severe lung lesions. In addition to that, HPAI also causes acute deaths as it also affects the brain inducing neuropathology and acute encephalitis. In our research endeavour, we are trying to establish and compare differences in the replication pathway (i) of H5N1 virus in three different host, and we chose to determine the transcriptomes regulated in three normal hosts that can be infected with this virus, that is the lungs from H5NI-infected chickens, primary chicken embryo lung cells and another mammalian tissue culture host i.e. Madin Darby Canine Kidney cells (ii) of two different viruses that cause lesions in the same organs or tissues. In this case we chose, H5N1 and Newcastle disease virus that cause neuropathology in the brains of infected chickens. The main aim here is to demonstrate whether brain neuropathology follow similar pathway in any viral infections through the regulation of its transcriptomes. We realized that through our preliminary studies, the host cellular responses in different organ and cell cultures and of different viruses that cause pathology in the same organs were substantially different from each other. This is demonstrated by the regulation of different sets of differentially expressed transcripts participating in viral replication in each of the experiments. (iii) Finally, we chose to study the protein-protein interactions of the viral nucleoprotein (NP) with cDNA of chicken lungs (main site of infection), in attempts to elucidate the mechanisms of H5N1 viral replication. We identified a novel interaction between H5N1 NP and chicken Aly/REF; a cellular RNA export adaptor protein. Mammalian two hybrid and molecular docking studies showed that NP from different subtypes also binds to both chicken and human Aly/REF. NP and Aly/REF co-localize primarily in the nucleus. siRNA knockdown of Aly/REF had little effect on the export of NP and other viral RNA as there were no significant reductions in virus yields in knockdown infected cells. Interestingly, knockdown of UAP56, another member of the TREX complex showed significant reduction in viral titer in influenza infected cells, suggesting the possible role played by this TREX complex in nucleocytoplasmic transport of viral mRNA. Another interesting finding from our yeast two hybrid screen was the interaction between NP and both the SRSF3 and 9G8. Again, we used mammalian two hybrid and molecular docking to show NP from different subtypes also binds to both chicken and human SRSF3 and 9G8. NP and both the SRSF3 and 9G8 also co-localizes primarily in the nucleus. siRNA knockdown of SRSF3 and 9G8 showed reduction in virus yields in infected cells, suggesting that they also may play a role in aiding viral RNA export. Overall, our data have contributed in understanding the complex interplay between the host and H5N1, specifically on the host cell responses during infection and also how the virus hijacks the host cellular machinery for its own survival.